Wat. Res. Vol. 24, No. 6, pp. 743-750, 1990 0043-1354/90 $3.00 + 0.

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Printed in Great Britain. All rights reserved Copyright © 1990 Pergamon Press pie

A COMPARATIVE STUDY OF THE N A T U R E OF

BIOPOLYMERS EXTRACTED FROM ANAEROBIC
A N D ACTIVATED SLUDGES
J. W. MOgGAN, C. F. FORSTER* and L. EVlSONt
School of Civil Engineering, Birmingham University, P.O. Box 363, Birmingham, England

(First received July 1989; accepted in revised form December 1989)

Abstract--The chemical nature of sludge extracellular polymers are widely reported as being influential
in determining many of the physico-chemical properties of sludges. A comparison was made between
biopolymers extracted from activated and anaerobic sludges including UASB granules. Noticeable
differences as to the total yield of ECP/(g SS) were observed. Activated sludge samples produced 70-90 mg
ECP/(g SS) compared with 10-20mg ECP/(gSS) for granular sludge. A relationship between the
concentration of ECP extracted and the surface charge of the sludge solids was observed. Activated sludges
were found, using a colloid titration technique, to be more electronegative than granular sludges. Gross
chemical analysis of these exti'acted polymers suggests that anaerobic an d activated sludge polymers do
differ, with protein being the most dominant fraction in anaerobic samples compared with carbohydrate
in the latter. The relative concentrations of elements present in the ashed extracts, measured by EDAX,
show a predominance of phosphorus and calcium. The actual concentrations of these two elements were
found to be greatest in the extracts from the anaerobic granules and anaerobic fluidised bed biofilm.

Key words--biopolymers, anaerobic digestion, USB granules, surface charge

INTRODUCTION
Extracellular polymers (ECPs) can be described
as being high molecular weight compounds
( M w > 10,000) produced by microorganisms under
certain environmental conditions. Such biopoiymers
originate from biological synthesis and excretion
or as lysis products from the lyric activity, pre-
dominantly of bacteria, and are usually found on floc
surfaces. These surface ECPs have previously been
referred to as mucilage and are consistent with the
glycocalyx described by Costerton and Irvin (1981).
This has been established to be an almost universal
surface component of the bacterial cell. The glyco-
calyx can be defined as any polysaccharide containing
structure, of bacterial origin, lying outside the outer
membrane of Gram negative cells and the peptido-
glycon of the Gram positive cell. Subsequently,
because of the position of these biopolymers, their
chemical composition affects the surface properties of
the bacterial flocs. ECP, therefore, is thought to affect
the physical properties of the sludge and is also
believed to be important in bioflocculation (Forster,
1971).
Bioflocculation is essential to enable the efficient
and economic operation of both anaerobic and
activated sludge wastewater treatment processes.
Agglomeration of the active biomass permits the
separation of the sludge flocs from the treated liquors
*To whom all correspondence should be addressed.
tPresent address: Department of Civil Engineering,
Newcastle University, England.
and their consequent retention within the process.
Although the precise mechanisms involved i n bio-
flocculation are not yet fully clarified, biopolymers
are believed to play a central role in the process. The
sorptive and colloidal destabilisation reaction in bio-
logical systems, such as yeast flocculation, antibody
and antigen reactions and the cohesion of tissue cells,
have long been thought to be influenced by naturally
occurring polymers (Busch and Stumm, 1968). Con-
sidering flocculation in biological wastewater treat-
ment processes specifically, Tenny and Stumm (1965)
proposed that polymers exposed on the microbial
surface may act to absorb and bridge between cell
surfaces and therefore initiate floc formation.
The precise function o f biopolymers in relation to
bioflocculation and their effect on sludge physico-
chemical characteristics, such as settlability, de-
watering, floc strength and charge, are incompletely
understood. However, the role attributed to extra-
cellular polymers with respect to the stabilisation of
colloidal particles is well established. Most of the
proposed mechanisms for bioflocculation are based
on the complex interactions between these high
molecular weight polymers which bond electro-
statically and physically to microbial surfaces. The
subsequent bridging that occurs between bacterial
cells and other particulate materials forms a heter-
ogenous matrix of such a magnitude as to allow
settlement of the floe aggregate. Such a mechanism is
widely reported in aerobic conditions and Ross
(1984) has suggested it could also be important to the
granulation process in anaerobic digesters.

743
WR 24/6~F
744 J.W. MORGANet al.
Biopolymers are also thought to be influential in
determining other sludge characteristics. The con-
centration and nature of such ionogenic polymers
present at sludge surfaces will determine the sludge
surface charge, which is usually negative having
values for activated sludge between - 10 to - 2 0 mV
(Horan and Eccles, 1986). Forster and Dallas-
Newton (1980) found that if the electronegativity of
the floc surface was sufficiently large, repulsion may
occur that would cause the sludge settling properties
to deteriorate. Also, an increase in sludge electro-
phoretic mobility was found by Magara et al. (1976)
to be related to increased polymer concentrations
which also worsened the settling characteristics of
the sludge. It seems feasible to presume, therefore,
that the chemical nature of the sludge surface will
influence the measurable floc charge which itself
affects the settling properties of the sludge. Addition-
ally the possible importance of ECP in the absorption
and subsequent removal of metal ions from solution
have also been reported (Brown and Lester, 1979).
Many attempts to extract and quantify sludge
biopolymers have been made because of their
involvement in bioflocculation. Several methods have
been developed for their extraction and characteris-
ation. These include steaming (Wallen and Davis,
1972), centrifugal stripping (Pavoni e t al., 1972),
alkali stripping (Sato and Ose, 1980) and ethanolic
extraction (Forster and Clarke, 1983). Amongst
others, Rudd et al. (1983) and Novak and Haugen
(1981) have compared the efficiency of these tech-
niques, using activated sludge, and found consider-
able variation in the relative abundance of the
polymer constituents, depending on the severity of
the method. This suggests that no universal method
exists, at present, to extract biopolymers quanti-
tatively and that comparison between different
authors' results must be made with caution. How-
ever, as suggested by Goodwin (1988), comparative
studies, such as the work described in this report,
can be useful if the extraction technique is initially
standardised.
Much of the qualitative studies relating to the main
chemical constituents of sludge biopolymers have
used activated sludge. These polymers have been
shown by Goodwin and Forster (1985) to be com-
posed of protein, carbohydrates, nucleic acids and
lipids. In the published literature, the ratio between
each of these component polymers varies, depending
on the sample source and extraction technique.
However, as suggested by Henning-Ryssov-Nielson
(1975), the polysaccharide component is most domi-
nant in activated sludge systems. Consequently, the
vast majority of research related to surface polymers
in activated sludge has been mainly concerned with
the carbohydrate fraction. The composition of bac-
terial extracellular polysaceharides has been exten-
sively reviewed by Sutherland (1985) who suggests
that polymers produced by certain bacterial species
are almost composed entirely of neutral sugars and a
limited number of uronic acids. Horan and Eccles
(1986) have shown that this is true for activated
sludge polymers. Analysis of the monomer compo-
sition of five purified sludge polysaccharides from
different treatment works shown that five monomers;
namely glucose, galactose, mannose, glueuronie acid
and galacturonic acid; were the most abundant by
percentage weight in all samples. Furthermore,
Forster (1971) showed that the principle ionogenic
component of such polysaccharides to be glucuronie
acid which supports this suggestion.
Published data related to the chemical nature of
anaerobic sludge surface polymers is less widely
reported in the literature. However, Karapanagiotis
et al. (1989) showed that, overall, anaerobically di-
gested sludge gave lower yields of ECP than activated
sludge, using similar techniques of extraction. Bowen
and Keinath (1984) and Forster (1982) also con-
cluded that digested sludge gave an overall lower
yield of extractable ECP. Although a study of ECP
from granular sludge has not been widely reported,
Ross (1984) found that such sludge gave an ECP yield
of 4% of the total dried sludge solids; considerably
less than that reported for activated sludge. Regard-
ing the chemical nature of sludge ECP, digested
sludge has been found to contain lipopolysaccharides
and proteins as the most significant components,
Forster (1983), rather than carbohydrates, as found
in activated sludge.
The aim of this paper is to present data to allow
a detailed comparison to be made between ECP
extracted from anaerobic and activated sludges. The
same standardised extraction techniques will be used
for each of the sludge samples, so as to minimise
the considerable variation previously reported in
the literature. The qualitative examination of ECP
extracted from granular sludge is in itself of im-
portance because of the lack of such reported infor-
mation. Such data could be useful to indicate the
possible role of ECP in the granulation process.
In addition, the relationship between the chemical
nature of the extractable polymer and other measur-
able parameters, including floc charge and overall,
structure will be made.
MATERIALS AND METHODS
Sludge samples
A comparative study was made between activated sludge,
digested sludge, upflow sludge blanket (UASB) granular
sludges (provided by Biwater Treatment Ltd and by
Davidson & Sons, Aberdeen) and samples from the SERC
anaerobic pilot-plant facility based at Gloucester (Forster,
1988). Details of the samples analysed in this investigation,
including the source and overall structure, are summarised
in Table 1.
All samples, with the exception of the granular sludges,
were received within 2 h after collection and stored in sealed
containers at 4°C. Measurements relating to the physical
nature of the sludges, including the initial stages of the
thermal extraction of the ECP, were completed within 2
days of sampling or, in the case of the commercial sludges,
of receiving the sample. A dried sample of ECP, extracted
 Biopolymers extracted from sludges 745

Table 1. Details of sludge samples used in the investigation

TSS
Sample Source (g/l) Sludge structure
Laboratory 10 litre continuously-fed 1.87 Flocculant
activated sludge reactor
(feed: raw sewage)
Kidderminster Activated sludge; Kidderminster 4.84 Floeculant
STW Treatment Plant
Bromsgrove Digested sludge, Bromsgrove 9.68 Looselyflocculant
STW reactor
Gloucester UASB reactor, Caernarvon 6.33 Looselyflocculant/dispersed
seed Creameries
(feed: dairy waste)
Gloucester 0.96 Dispersed
UASB
SERC anaerobic pilot plant
Gloucester facility sited at Gloucester 0.86 Dispersed
anaerobic filter (feed: waste ice cream)
Gloucester 0.95 Dispersed
fluidised bed
Aberdeen UASB (Paques), Davidson & 102.67 Granular
granules Sons Papermill, Aberdeen
(feed: paperwaste water)
Dutch UASB (Paques). Provided by 98.51 Granular
granules Biwater Treatment Ltd
U.K. granules Lab-scale UASB, University Dried Granular
Birmingham, grown from ECP
Bromsgrove DS

from granular sludge grown from digested sludge in
Birmingham, was also analysed.
Determination of sludge and ECP surface charge
Measurement of the sludge surface charge was accom-
plished using the colloid titration technique described by
Kawamura and Tanaka (1966). All solutions were adjusted
to neutrality prior to titration. Polybren and polyvinyl
sulphate (PVSK) were used as the standard cationic and
anionic colloids, as suggested by Tiravanti et al. (1985). A
known volume of the sample was diluted in distilled water
and mixed with excess 0.001 N Polybren then titrated
against 0.001 N PVSK until electrical neutrality was
reached. A few drops of Toluidine blue added before
titration were used to indicate this end point; a subtle colour
change from blue to pink/purple. Equal volumes of Poly-
bren in distilled water were used as blanks. The colloid
charge expressed as milliequivalents per litre of positive or
negative colloid charge can then be determined from the
expression given below. For sludge samples it is preferrable
to express this charge per gram of dried sludge solids.
A - B N (1000)
Charge (mequiv/l)
V Standards of bovine serum albumen (BSA) within the range
where
A= ml of PVSK added to the sample
N= normality of PVSK
B= ml PVSK added to blank
V= ml sample used.
Extraction of sludge extracellular polymers
The thermal extraction/solvent precipitation techniques
described by Forster and Lewin (1972), Brown and Lester
(1980) and Forster (1971) was used to extract the polymeric
material from all the sludges. A measured volume of sludge
solids was concentrated by low speed centrifugation
(2000 rpm) and resuspended in 1/4 strength Ringers sol-
ution. The concentration of dried sludge solids was then
determined by filtration of a sample of this suspension. To
reduce bacterial lysis and consequent release of intracellular
products, thermal treatment at 80°C was used as suggested
by Goodwin (1988).
The extracted polymers were harvested by removal of the
sludge solids separated by centrifugation first at 2000 rpm
and then at 10,000 rpm. The ECP in the supernatant was
precipitated using a solvent mixture of acetone and ethanol
(3 vol: 1 vol) and left overnight. The precipitate was subse-
quently removed from the solvent by centrifugation and was
dehydrated using acetone and petroleum ether rinses before
determining the yield.
Qualitative analysis of extracted ECP
Samples of dried extracts were dissolved in water to give
a 1 g/1 solution which was then filtered through a 0.45 #m
membrane. The total organic carbon protein and carbo-
hydrate concentrations were then determined.
The measurement of total organic carbon (TOC) was
performed using a Beckman 915 TOC analyser connected to
the Beckman 215B infra-red analyser. 30#1 Samples were
used for all measurements. Carbohydrates were determined
using the phenol/sulphuric acid method of Dubois et al.
(1956). The colour obtained was allowed to develop for
30min before the absorbance read against a blank at
490 nm. Glucose was used as the standard in the range
0-100 mg/1. Proteins in the extracts were determined using
the modification of the Folin/Ciocalteu method described by
Lowry et al. (1951). The absorbance of the colour which was
developed after 30 min was read at 750 nm against a blank.
0~0mg/1 were used to determine the concentration of
crude proteins in the ECP. As in previous studies, neither
the glucose standards nor the BSA were subjected to
the extraction procedures. However, the consistency of the
analytical methods was checked by measuring the carbo-
hydrate and protein concentrations in polymer solutions to
which known concentrations of glucose and BSA had been
added. In addition, two of the ECP samples were subjected
to amino acid analysis. These analyses were done under
contract by Alta Bioscience, Birmingham.
The elements present in the polymer extracts after being
ashed at 500°C were determined using electron dispersive
X-ray analysis (EDAX). Samples of the dehydrated ash
were attached to graphite slabs with a carbon based
adhesive and examined using a Joel JEM 100 CX2 EM
AND LINK 860 series 2 (Link systems) EDAX analyser.
Five randomly selected areas were scanned and the
combined spectra used to determine the mean relative
percentage of the most predominant elements.
Determination of the actual concentrations of calcium in
the extracts was achieved using flame photometry. All the
samples were diluted in distilled water to give a final
746 J.W. MORGANet al.

Table 2. Colloidalsurface charge of solids and ECP 100

Charge on Charge on
sludge solids extractedECP /
Sample (mequiv/gSS) (mequiv/gECP) 80 /
Lab AS --0.683 --0.910
KidderminsterAS --0.895 --0.50
Bromsgrove DS - 0.110 - 0.632 x~ 60 ,/
Gloucester seed -0.570 -0.91
Gloucester UASB -0.720 -0.80
Gloucester FB -0.907 -0.73
Gloucester AF -0.483 -0.93 iii 40 ...... "- •
Aberdeen granules -0.260 -0.203
Dutch granules --0.430 --0.375
20
concentration of 1 mg/1 of extract. The flame photometer
(model A, Evans Electroselenium Ltd) was used with the f i '¢ I I ~ I
appropriate filter fitted. Standards within the range -0"2 -0"4 -0'6 -0"8 -1"0
0-100mg/1 Ca were prepared from a stock solution of
Surface charge (meq/g)
CaCO3. A calibration curve of calcium concentration
against absorbance was used to determine the amount of Fig. l. Relationship between sludge surface charge and the
calcium in the extracts. biopolymer yield, showing the regression line and the 95%
envelope.
RESULTS
The Gloucester samples from the fluidised bed, the
Surface colloid charge of sludge solids and extracts anaerobic filter and the UASB reactors yielded inter-
The colloid titration technique proved to be a mediary amounts of ECP compared with the acti-
reproducible method to determine the surface charge vated sludge and granular sludges.
of sludge solids. Initial experiments show that if the A relationship between the concentration of ex-
pH and the temperature of titration were kept con- tracted ECP and the charge carried on the sludge
stant, the standard deviation of the calculated charge solids was established (Fig. 1); the higher yields of
remained low for the same sample. The calculated extract correlating with the greater values of the
colloidal surface charge for the sludge solids and their overall electronegativity of the sludge surface. Acti-
extracts shown in Table 2, are all expressed as vated sludge samples yielded more ECP than any of
milliequivalents per gram of sludge solid and ECP the anaerobic sludges and also had a more negatively
respectively. As expected, all the sludge solids and charged sludge surface. This suggests that the amount
their extracts carry an overall negative charge, how- of ECP may be responsible for determining the
ever, the charge carried by both the granular sludges surface charge characteristics of the sludge.
and their ECP extracts was found to be less negative The concentrations of TOC, protein and carbo-
than that of the activated sludges examined. Interest- hydrate determined in the dried ECP extracts are
ingly, the charges carried by the more dispersed shown in Table 3. This table also shows the ash
Gloucester samples were more similar to those of the content of the ECP and the amount of polymeric
activated sludge than the digested or granular material that remains unidentified. This unclassified
sludges. fraction could be composed of lipids or nucleic acids.
Both types of compound have previously been re-
Yields of extracted extracellular polymers ported in sludge ECP (Magara and Nambu, 1976;
The total yields of extracted ECP were calculated Forster, 1982; Robinson et al., 1984). Results from
from the dried weight of the precipitated material the TOC analysis show that, for all the samples
removed after thermal treatment at 80°C for 1 h. analysed, no measureable concentration of inorganic
From the summarised results, shown in Table 3, it carbon was detected indicating that the carbon
appears that the two activated sludge samples contain content of the ECP extracts was composed almost
more extractable ECP than the anaerobic sludges. entirely of organic carbon. The activated sludge

Table 3. Concentrationsof extractable materials

Yield
Total Yield carbohydrate Yield protein Unidentified
yield TOC (glucose equiv) (BSA equiv) Ash component
Sample (mg/g SS) (mg/g SS) (mg/g SS) (mg/g SS) (% ECP) (% ECP)
Lab AS 69.9 18.38 13.98 11.39 34.5 29.0
KidderminsterAS 90.2 27.51 23.90 14.07 18.0 40.0
Bromsgrove DS 13.3 3.49 1.93 5.39 35.1 1.0
Gloucester seed 16.3 5.54 1.87 3.85 -- --
Gloucester UASB 46.8 12.30 8.42 9.01 -- --
Gloucester AF 25.7 6.76 3.98 5.44 -- --
Gloucester FB 44.I 14.64 6.39 8.49 -- --
Aberdeen granules 10.4 3.18 2.34 2.90 25.4 24.2
Dutch granules 19.5 5.03 4.29 5.36 11.2 39.2
 Biopolymers extracted from sludges 747

Table 4. Ratio of extracted protein:carbohydrate 3.0

Ratio of • x
Sample protein:carbohydrate
Lab AS 0.85 2.5 "xx
KidderminsterAS 0.59 (0.59)*
Bromsgrove DS 2.79
Gloucester seed 2.05 m 2.0 ". •
Gloucester UASB 1.09
Gloucester AF 1.37
Gloucester FB 1.33 "o
Aberdeen granules 1.24 (1.09)* 1.5
Dutch granules 1.25 o
..Q

*Protein based on amino acid analysis.

•.
t-
1.0

samples appeared to give higher concentrations of ~- 0.5 "', •

TOC than the granular and digested sludges. Com-
parison between the yields of carbohydrate and
protein suggest a difference between the crude chemi-
cal nature of ECP extracted from the aerobic acti-
vated sludge and anaerobic sludges. As previously
suggested by Forster 0983), the protein component
in anaerobic sludges appeared to be the most sig-
nificant, whereas the carbohydrate component was
most dominant in activated sludge. This difference is
more clearly evident when the ratios of extractable
protein:carbohydrate are compared (Table 4). These
ratios can also be correlated with the surface charge
(Fig. 2).
The relative percentage of the most abundant
elements detected by E D A X analysis and a typical
EDAX spectrum are shown in Fig. 3. All the sludge
samples irrespective of their source were found to
contain two predominant elements, phosphorous and
calcium. Although the proportions of these elements
did vary, in all samples more than 85% of the
detectable elements could be accounted for by phos-
phorous and calcium. In addition variable quantities
of Na, Mg, Al, Si, S, C1, K and Fe were present in
the ashed extracts.
From the concentration of calcium in the extracts,
determined using flame photometry, the concen-
I / 1 t I
-0.2 -0.4 -0"6 -0.8 -1.0
Surface charge (meq/g)
Fig. 2. Relationship between the sludge surface charge
and the protein:carbohydrate ratio in the extracted ECP,
showing the regression lines and the 95% envelope.
tration of the other elements detected using E D A X
were calculated from the relative percentages. These
results (Table 5) revealed that higher concentrations
of all elements, in particular calcium and phos-
phorous, are present in the ashed extracts from the
granular sludges and the fluidised bed biofilm.
DISCUSSION
The yield of extracted polymeric material was
found to differ significantly depending on the nature
of the sludge sample. All the anaerobic samples, in
particular the digested and granular sludges, yielded
significantly less ECP than the activated sludges. This
is compatible with earlier data (Table 6) and may
indicate that under anaerobic conditions fewer bio-
polymers are released, a suggestion supported by

Aberdeen granules

8O

70

6O

5O

40

"50

10--
0/ ~a ' ~ ' P ' $ ' CL ' K ' C
ELemenLs
Fig. 3. Typical EDAX spectrum for ashed ECP.
748 J . W . MORGAN e t al.

Table 5. Concentration of elements in the ashed ECP extracts (determined from the relative percentages from EDAX and the [Ca]
determined by flame photometry)
-- Concentration of the predominant elements in the ashed extracts
(#g/g ECP)
Sample Na Mg A1 Si P S CI K Ca Fe
Lab. AS 3.45 24.12 0.00 0.00 937.30 0.00 0,00 31.01 1998.65 99.93
Kidderminster AS 5.60 31.72 0.00 0.00 535.45 0.00 0.00 16.79 529.85 16.79
Lab. FB 320.57 606.57 44.00 1084.29 5286.29 0.00 1015.14 72.29 5333.43 157.14
Bromsgrove DS 3.51 10.53 3.51 0.00 610.53 7.02 35.10 52.63 2340.35 3.51
Aber gran. 30.45 154.78 27.91 0.00 4617.91 0.00 17.76 20.30 5074.63 0.00
Dutch gran. 224.72 591.01 15.73 0.00 6267.42 0.00 46.07 475.28 2234.83 19.10

Table 6. Comparison of the ECP yields obtained from different
sludges
Source Sludge type
Kiff (1978) Activated
This study Activated
Clarke and Forster (1982) Activated
Forster and Clarke (1983) Activated
Karapanagiotis et al. (1989) Digested
Dolfing (1986) Digesting granules
This study Various digesting
sludges
Karapanagiotis et aL (1989). Alternatively, Henning-
Ryssov-Nielsen (1975) showed that, under anaerobic
conditions, bacterial biopolymers may be quickly
degraded by the bacteria, forming COs and CH4 as
byproducts. If true, this could explain why lower
concentrations are present in systems where the
availability of oxygen is limited.
The analysis of the activated sludge ECP indicates
that the dominant component is a carbohydrate;
Pavoni et al. (1972) also found this to be true.
However, the ratio between the protein and carbo-
hydrate components has previously been found to be
variable, depending on the extraction method and the
course of the sample. The type of waste being treated
is of particular relevance. For example, Vallom and
McLoughlon (1984) found that the protein fraction
was the most prevalent in ECP extracted from acti-
vated sludge that had been acclimatised to a feed
containing high concentrations of BSA.
However, in general, anaerobic sludges tend to
have higher concentrations of protein in their ex-
tracted polymers (see Table 7). The prevalence of pro-
tein fractions has been found previously by Forster
(1983) and by Karapanagiotis et al. (1989) who
quoted protein:carbohydrate ratios of approx. 3 : 1
for digested sludge ECP; a result similar to that
reported in this investigation. The protein:carbo-
hydrate ratios showed a linear relationship with the
surface charge, with the correlation coefficient being
ECP yield
(% TSS) 0.66 (which was significant at the 95% level). These
6.47
differences between the gross chemical nature of
8.00 aerobic and anaerobic biopolymers suggests that in
4.7 7.2 the latter systems the different metabolic processes
10.8
3.5 result in the production of polymers which not only
2 are released less readily but are also chemically
2.5 different.
A relationship between the concentration of sur-
face biopolymers and the sludge surface charge was
also established. The correlation coefficient for this
relationship was 0.66 which was just significant at the
95% level. Magara et al. (1976) have demonstrated
the influence that increased biopolymer synthesis had
in increasing the surface charge, measured by in-
creased electrophoretic mobility. High concentrations
of anionic surface biopolymers can consequently be
correlated with deteriorating sludge settling charac-
teristics because of the influence of floc-repulsion.
Bearing this in mind, it is interesting to note that the
highly dispersed samples from the Gloucester SERC
pilot-plant carried relatively high negative charge
compared with granular sludge solids and ECP
extracted from the granules.
The mineral composition of anaerobic granular
sludge has previously revealed the presence of
high concentrations of Ca, Mg, K, Fe, S and P
(Dubourguier et al., 1988). When the elemental com-
position of the surface alone was examined by
Dubourguier et al. (1988), a predominance of phos-
phorous, similar to that in the extracted ECP found
in this study, was detected. Such high concentrations
of this element in the ashed polymer extracts may
possibly originate from the phosphate groups abun-
dant in biopolymers such as phospholipids and
nucleic acids. Calcium was also found to be an
important constitutent of the ashed extracts from

Table 7. Comparison of the protein:carbohydrate ratios in sludge ECPs

Source Sludge type Protein:carbohydrate
Horan and Eccles (1986) Activated 0.16
Forster and Clarke (1983) Activated 0.56
This study Activated 0.70
Karapanagiotis et al. (1989) Digested 3.0-3.6
Forster (1982) Digested 5. I
Ehlinger et aL (1987) Digested
(a) Glucose fed 0.61
(b) VFA fed 2.31
This study Various digesting 1.1-2.8
sludges
 Biopolymers extracted from sludges
all the samples studied. In anaerobic systems, this
element is commonly deposited as the carbonate
which is precipitated in zones where CO2 is being
produced by microcolonies of methanogens. How-
ever, because of the high levels of phosphorous, it
seems probable that calcium phosphate is also pre-
cipitated and indeed such deposits were detected by
Dubourguier et al. (1988) by elemental mapping of
granules.
Bivalent cations, in particular calcium, have been
implicated in the bacterial aggregation process be-
cause of their ability to bridge between the electro-
negative carboxyl and phosphate groups associated
with bacterial surfaces. The high concentration found
in all samples may be due to the binding of the
calcium ion between these anionic groups on the
biopolymers. Calcium may also be influential to
the stabilisation of bacterial conglomerates but an
attempt by Hulshoff-pol et al. (1986) to relate the
strength of granules to their mineral content was not
conclusive. Also, Guiot et al. (1988) have shown that
calcium did not seem to be a key factor involved in
inducing granulation. However, the high concen-
tration of this element in granular sludge may
contribute to the overall stability and possibly
be responsible for reducing the measured surface
negativity of such sludges.
It is possible that the production of exopoly-
saccharides is restricted in methanogenic bacteria. If
true this could explain why anaerobic sludges such as
stable granules that possess a strong population of
methanogens yield less ECP. Unfortunately extra-
cellular polymer synthesis in these bacteria has not
been studied in any detail. However, unlike most
procaryotes, methanogens do not possess peptido-
glycan in their cell walls. Instead, most methanogens
consist of either a polypeptide sacculus or outer
proteinacous sheath (Kandler and Konig, 1978;
Sprott and McKellar, 1980). One essential com-
ponent to peptidoglycan synthesis is a lipid carrier
molecule (undecaprenyl phosphate) which is involved
in transporting the peptidoglycan precursors across
the bacterial cytoplasmic membrane. This lipid
carrier has also been implicated in the release
of exopolysaccharides and capsule formation
(Hammond et al., 1984). Therefore, in methanogens,
this component may be missing and so exopoly-
saccharide in addition to peptidoglycan synthesis
would be limited. In this context it is interesting that
methanogens are less sensitive to antiboiotics such as
vancomycin and penicillins which interfere with the
synthesis of peptidoglycan and presumably other
bacterial polymers (Alexander et al., 1980).
The role of high molecular weight polymers in
adhesion and the stabilisation of the resulting biofilm
are well established. Although the mechanisms in-
volved in the initiation of granulation are not fully
understood the probable involvement of certain func-
tional polymers has been suggested (Ross, 1984). The
functional polymers need not necessarily be poly-
749
saccharide in nature for proteins may provide the
positive groups which form bonds between the nega-
tive sites on the bacteria. Other compounds including
lipopolysaccharides have also been found in the
extracellular matrix of anaerobic biofilms (Robinson
et al., 1984). The concentration of biopolymers is
not intimately important to the stabilisation of
the biofilms because a large number of contacts can
exist between these and the bacteria. Probably more
important is the arrangement of functional groups
exposed along the surface of the polymers.
If indeed bacterial ECPs are important, a more
detailed study regarding their chemistry may help in
the understanding of the process. However, work
relating to the chemical nature of granular sludge
polymers is not widely reported in the literature.
From this study, one can conclude that the low
concentrations of ECP and the subsequently reduced
surface negativity are important to the stability of the
very compact structures which constitute granular
sludge.
Acknowledgements--The authors gratefully acknowledge
the support of SERC (Grant No. GR/E 2873.4), Unilever
Research and the staff of the Birds Eye-Walls factory at
Gloucester.
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